Adaptation to serum-free culture of HEK 293T and Huh7.0 cells

Paschoal, Juliana Fontes Beltran; Patiño, Sandra Suarez; Bernardino, Thaissa Consoni; Rezende, Alexandre Gonçalves de; Lemos, Marcos Alexandre Nobre; Pereira, Carlos Augusto; Jorge, Soraia Attie Calil

doi:10.1186/1753-6561-8-s4-p259

Cited by 5 publications

(5 citation statements)

References 3 publications

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“…In addition, there is a risk of contamination with bovine viruses or mycoplasma that could affect the results and reproducibility in vitro cell culture system ( Groothuis et al, 2015 ; Baker, 2016 ; van der Valk et al, 2018 ). Therefore, the production of vaccines and therapeutic proteins in the biopharmaceutical industry and biotechnology products for clinical purposes should be prepared in serum-free culture systems ( Beltran Paschoal et al, 2014 ; Miki and Takagi, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, going from serum-containing to serum-free cultures requires adaptation steps that progressively reduce serum concentration in the medium ( Doyle and Griffiths, 1998 ; Biaggio et al, 2015 ). A key strategy for achieving successful serum-free culture is that the procedure provides sufficient time to adapt to the new medium conditions and those changes are gradually introduced ( Beltran Paschoal et al, 2014 ). Nevertheless, the transition to serum-free culture can be challenging, labor-intensive, and time-consuming ( Brunner et al, 2010 ; Caron et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

“…Its ability to grow in suspension culture in different commercial media has been reported. ( Beltran Paschoal et al, 2014 ; Lomba et al, 2021 ). While HEK293 metabolism related to the vaccine or therapeutic protein production was extensively explored ( Petiot et al, 2015 ), the information regarding the potential metabolic effects of serum-free culture and suspension growth is scarce.…”

Section: Introductionmentioning

confidence: 99%

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The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

Jang

Pete

Bruheim

2022

Front. Bioeng. Biotechnol.

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Serum-free cultures are preferred for application in clinical cell therapy and facilitate the purification processes of bioproducts, such as vaccines and recombinant proteins. It can replace traditional cell culture - eliminating potential issues posed by animal-derived serum supplementation, such as lot to lot variation and risks of pathogen infection from the host animal. However, adapting cells to serum-free conditions can be challenging and time-consuming, and is cell line and medium dependent. In addition, the knowledge of the impact of serum-free culture on cellular metabolism is limited. Herein, we successfully established serum-free suspension and adherent cultures through two adaptation procedures for HEK293 cells in serum-free Freestyle 293 medium. Furthermore, growth kinetics and intracellular metabolic profiles related to central carbon metabolism were investigated. The entire adaptation procedure took 1 month, and high cell viability (>90%) was maintained throughout. The serum-free adherent culture showed the best growth performance, measured as the highest cell density and growth rate. The largest differences in metabolic profiles were observed between culture modes (adherent vs. suspension), followed by culture medium condition (control growth medium vs. serum-free medium). Metabolic differences related to the adaptation procedures were only seen in suspension cultures. Interestingly, the intracellular itaconate concentration was significantly higher in suspension cells compared to adherent cells. Furthermore, when the cells back-adapted from serum-free to serum-supplemented control medium, their metabolic profiles were immediately reversed, highlighting the effect of extracellular components on metabolic phenotype. This study provides strategies for efficient serum-free cultivation and deeper insights into the cellular responses related to growth and metabolism responses to diverse culture conditions.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

Jang

Pete

Bruheim

2022

Front. Bioeng. Biotechnol.

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“…Afterward, cells with shaking were sampled and counted every generation. The adaption criteria for 293 T suspension cells were the cell density from 4.0 × 10 5 cells/mL to 1.6 × 10 6 cells/mL after 3 days in culture, while cell viability of more than 90% was maintained [ 9 , 19 ].…”

Section: Methodsmentioning

confidence: 99%

“…The trend significantly improves the transition from laboratory to clinical-grade production. A previous study reported that the 293 T cell had been adapted in serum-free media as suspension cells using sequential adaptation approach [ 9 ]. However, using this approach with a long adaptation cycle [ 10 ], the proliferation of 293 T suspension cells was slower than original adherent cells, also LV yield.…”

Section: Introductionmentioning

confidence: 99%

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy

Tang

et al. 2021

Bioengineered

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As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serumdependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 10 7 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

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Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model

et al. 2018

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The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco’s Modified Eagle’s Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

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Adaptation to serum-free culture of HEK 293T and Huh7.0 cells

Cited by 5 publications

References 3 publications

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model

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